The process of biological hydroxylation as accomplished by mammalian liver microsomal cytochrome P-450 will be scrutinized. The general objective will be to determine the detailed mechanism of oxygen activation and insertion into substrate. Two modes of oxygen activation are catalyzed by cytochrome P-450, one utilizing NADPH/O2 and the other utilizing organic hydroperoxides. Specific aims are to determine chemical identities for the activated oxygen intermediates in both modes and to establish the mechanistic relation between the two. A new mechanistic proposal, the quasi-Fenton mechanism, will be tested in this project. Longer range goals include the development of a predictive model for the oxidation of xenobiotics and a general theory of heme-oxygen chemistry. Purified, homogeneous cytochrome P-450 will be prepared and used. The basic experimental methods will be the inference of chemical characteristics of the activated oxygen from its behavior toward selected substrates. The approach will be that of physical organic chemistry and specific experiments will seek to determine the presence of radicals, internal hydrogen isotope selectivity, stereochemistry, and the chemical characteristics of the activated oxygen.